Hypoxia or oxygen tension below normal physiologic levels in cells results in physiologic as well as pathologic alterations in the cells, which alterations have been associated with differential gene expression. For example, hypoxia affects endothelial cellular physiology in vivo and in vitro in various ways including modulating the transcriptionally-regulated expression of vasoactive substances and matrix proteins involved in modulating vascular tone or remodeling the vasculature and surrounding tissue (Faller, D. V. Clin. Exp. Pharmacol. and Physiol. 1999 26:74-84). Hypoxia in solid tumors has been shown to protect cancer cells from being killed by X-irradiation and leads to resistance to certain cancer drugs. Hypoxia also appears to accelerate malignant progression and increase metastasis (Brown, J. M. Cancer Res. 1999 59:5863-5870).
Low oxygen levels have been shown to enhance invasiveness in a number of cell types, and likely promote invasive phenotypes in biological environments such as the core of rapidly growing tumors. Studies in our laboratory suggest that this effect is mediated via a reduction in nitric oxide (NO) production. For example, the inhibition of endogenous NO production (that occurs under hypoxic conditions or in the presence of a NOS inhibitor) was shown to up-regulate both the expression of the urokinase receptor (uPAR, a pro-invasive molecule) and metastasis. Further, the hypoxic upregulation of (uPAR) and metastasis was completely abrogated by the addition of very low concentrations of NO-mimetics.
Nitric oxide has been implicated in various biological processes. For example, nitric oxide is a biological messenger molecule responsible for endothelium derived vascular relaxation and neurotransmission. Nitric oxide, at what these researchers refer to as high levels, is also known as a mediator for anti-tumor and anti-bacterial actions of macrophages. Nitric oxide has also been demonstrated to play a modulatory role on cytokine-induced expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases (Eberhardt et al. Kidney International 2000 57:59-69).
Soluble guanylyl cyclase (sGC) is the best defined downstream target of NO. However, since NO is a pleiotropic molecule, it is likely that there are other molecular targets that mediate NO action. For example, it is very likely that NO could attenuate gene expression via currently unknown mechanisms and, as such, impact various cellular functions, such as growth, proliferation, migration, apoptosis, and the like. The heterodimeric protein, i.e., sGC, catalyzes the conversion of guanosine 5′-triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). Nitric oxide binds to the heme moiety of sGC, inducing a number of conformational changes that result in its activation, cGMP is a potent second messenger that allows NO to transmit its signal to downstream effectors. Elevated levels of cGMP have been negatively correlated with vascular smooth muscle growth and have been shown to prevent platelet aggregation as well as the adherence of neutrophils to endothelial cells. Further, cGMP levels play a role in gene regulation. A cGMP analogue (8-Br-cGMP) has been shown to prevent the hypoxic induction of VEGF. The endogenous inhibition of endothelin-1 (ET-1) by NO is also mediated through a guanylate cyclase/cGMP-dependent mechanism.
There are several cGMP target proteins that could mediate NO-regulated gene expression. These include cGMP-dependent protein kinase (PKG), cGMP-activated phosphodiesterases (PDEs), and cGMP-gated ion channels, and under certain conditions, cAMP-dependent protein kinases. Of these, it is thought that PKG is responsible for the majority of cGMP's intracellular actions. PKG is a serine/threonine protein kinase that is selectively activated by cGMP binding. Upon activation, PKG phosphorylates many intracellular targets, often resulting in alterations in gene expression. For example, PKG has been shown to modulate the transcription of various genes through its activation of factors such as AP-1 and the serum response element. Similarly, Idriss et al. has shown that the c-fos promoter is activated by a PKG dependent mechanism.
Clinical and experimental data suggest that nitric oxide plays a role in promoting solid tumor growth and progression. For example, nitric oxide generation by inducible nitric oxide synthase (iNOS) has been implicated in the development of prostate cancer (Klotz et al. Cancer; National Library of Medicine, MDX Health Digest 1998 82(10):1897-903), as well as in colonic adenocarcinomas and mammary adenocarcinomas (Lala, P. K. and Orucevic, A., Cancer and Metastasis Reviews 1998 17:91-106). In addition, nitric oxide has been suggested to play an important role in the metabolism and behavior of lung cancers, and in particular adenocarcinomas (Fujimoto et al. Jpn. J. Cancer Res 1997 88:1190-1198). In fact, it has been suggested that tumor cells producing or exposed to what these researchers refer to as low levels of nitric oxide, or tumor cells capable of resisting nitric oxide-mediated injury undergo a clonal selection because of their survival advantage (Lala, P. K. and Orucevic, A. Cancer and Metastasis Review 1998 17:91-106). These authors suggest that these tumor cells utilize certain nitric oxide-mediated mechanisms for promotion of growth, invasion and metastasis and propose that nitric oxide-blocking drugs may be useful in treating certain human cancers. There is also evidence indicating that tumor-derived nitric oxide promotes tumor angiogenesis as well as invasiveness of certain tumors in animals, including humans (Lala, P. K. Cancer and Metastasis Reviews 1998 17:1-6).
However, nitric oxide has been reported to reverse production of vasoconstrictors induced by hypoxia (Faller, D. G. Clinical and Experimental Pharmacology and Physiology 1999 26:74-84). In addition, the nitric oxide donors sodium nitroprusside, S-nitroso-L-glutathione and 3-morpholinosydnonimine in the micromolar range (IC50=7.8, 211 and 490 μM, respectively) have been demonstrated to suppress the adaptive cellular response controlled by the transcription factor hypoxia-inducible factor-1 in hypoxically cultured Hep3B cells, a human hepatoma cell line (Sogawa et al. Proc. Natl. Acad. Sci. USA 1998 95:7368-7373). The nitric oxide donor sodium nitroprusside (SNP; 150 μM) has also been demonstrated to decrease hypoxia-induced expression of vascular endothelial growth factor, an endothelial cell mitogen required for normal vascular development and pathological angiogenic diseases such as cancer and iris and retinal neovascularization (Ghiso et al. Investigative Opthalmology & Visual Science 1999 40(6):1033-1039). In these experiments, 150 μM SNP was demonstrated to completely suppress hypoxia-induced VEGF mRNA levels for at least 24 hours in immortalized human retinal epithelial cells.
High levels of nitric oxide, when induced in certain cells, can cause cytostasis and apoptosis. For example, Xie et al. have demonstrated exposure to high levels of nitric oxide (producing approximately 75 μM nitrite; see FIG. 5A of Xie et al.) to be an exploitable phenomenon to promote death (see FIGS. 6A and 6B of Xie et al.) in murine K-1735 melanoma cells (J. Exp. Med. 1995 181:1333-1343). In addition, WO 93/20806 discloses a method of inducing cell cytostasis or cytotoxicity by exposing cells to a compound such as spermine-bis(nitric oxide) adduct monohydrate at 500 μM which is capable of releasing nitric oxide in an aqueous solution. The compounds are taught to be useful in the treatment of tumor cells as well as in antiparasitic, antifungal and antibacterial treatments. Use of a mega-dosing regimen is suggested, wherein a large dose of the nitric oxide releasing compound is administered, time is allowed for the active compound to act, and then a suitable reagent such as a nitric oxide scavenger is administered to the individual to render the active compound inactive and to stop non-specific damage. It is taught at page 14, line 25-30 of WO 93/20806 that 3-(n-propyl amino)propylamine bis(nitric oxide) adduct, diethylamine-bis(nitric oxide) adduct sodium salt, isopropylamine-bis(nitric oxide) adduct sodium salt, sodium trioxodinitrate (II) monohydrate, and N-nitrosohydroxylamine-N-sulfonate did not significantly affect cell viability at concentrations up to 500 μM.
U.S. Pat. No. 5,840,759, U.S. Pat. No. 5,837,736, and U.S. Pat. No. 5,814,667, disclose methods for using mg/kg quantities of nitric oxide releasing compounds to sensitize hypoxic cells in a tumor to radiation. These patents also disclose methods of using the same nitric oxide-releasing compounds at mg/kg levels to protect non-cancerous cells or tissue from radiation, to sensitize cancerous cells to chemotherapeutic agents, and to protect non-cancerous cells or tissue from chemotherapeutic agents. Compounds used in these methods spontaneously release nitric oxide under physiologic conditions without requiring oxygen. These patents teach administration of the nitric oxide-releasing compound from about 15 to about 60 minutes prior to therapy. Typical doses of the nitric oxide releasing compound administered are suggested to be from about 0.1 to about 100 mg of one or more nitric oxide releasing compounds per kg of body weight. Concentrations of the nitric oxide releasing compounds DEA/NO and PAPA/NO demonstrated to increase the sensitivity of MCF7 breast cancer cells and V79 fibroblasts to melphalan, thiotepa, mitomycin C, SR4233 and cisplatin in vitro were in the millimolar range while 70 mg/kg of DEA/NO was demonstrated to increase the survival of mice administered the chemotherapeutic agent melphalan in the in vivo KHT tumor model.
U.S. Pat. No. 5,700,830 and WO 96/15781 disclose methods for inhibiting adherence between cancerous cells and non-cancerous cells in an animal by administering to the animal a nitric oxide-releasing compound containing a nitric oxide-releasing N2O2 functional group. However, studies indicate that cancer cell adhesion to and spreading along the vessel wall leading to extravasation is not an obligatory event in metastasis (Morris et al. Exp. Cell. Res. 1995 219:571-578).
WO 98/58633 discloses a microdose nitric oxide therapy for alleviating vascular conditions associated with a reduction in nitric oxide production or an attenuation of nitric oxide effect.